Possible Involvement of Hepatic Phosphatidate Phosphohydrolase in the Mechanisms of Actions of Certain Antilipemic Drugs in Rats

The effects of therapeutic doses of dillsun, garsin, antum and statins on rat liver cytosolic phosphatidate phosphohydrolase (PAP) activity, a key enzyme in triacylglycerol synthesis, and on serum and liver lipids were examined. Lovastatin and simvastatin both stimulated the enzyme activity by 29% and 43%, respectively. The stimulatory effects were dose-dependent and accompanied by the decline in triacylglycerol and cholesterol concentrations of serum (20-29%) and liver (12-29%) suggesting a possible feed-back mechanism for PAP control. Dillsun (0.5-2 ml/day) did not affect PAP activity and liver lipids but lowered serum triacylglycerol (27%) and cholesterol (20%) concentrations at a dose of 2 ml/day. Antum administration also showed similar pattern as dillsun except that it caused a decrease in the liver lipids as well. Garsin (250-1000 mg/Kg) inhibited PAP activity (14-22%) with simultanous decrease in the liver and serum lipids; maximum decrease in serum triacylglycerol and cholesterol were 14% and 21%, respectively. The data demonstrated that the antilipidemic drugs tested exert their effects, at least in part, through alteration of hepatic PAP activity.

Copyright ? 2007 by School of PharmacyShaheed Beheshti University of Medical Sciences and Health Services

Original Article

Possible Involvement of Hepatic Phosphatidate Phosphohydrolase in the

Mechanisms
of Actions of Certain Antilipemic Drugs in Rats

Bahram Haghighi*, Mani Kharazizadeh and Mohammad Ali Attar

Deapartment of Clinical
Biochemistry, School of Pharmacy, Medical Sciences University of Esfahan,
Esfahan, Iran.

Abstract

The effects of therapeutic doses of dillsun, garsin, antum and statins on rat
liver cytosolic phosphatidate phosphohydrolase (PAP) activity, a key enzyme in
triacylglycerol synthesis, and on serum and liver lipids were examined.
Lovastatin and simvastatin both stimulated the enzyme activity by 29% and 43%,
respectively. The stimulatory effects were dose-dependent and accompanied by the
decline in triacylglycerol and cholesterol concentrations of serum (20-29%) and
liver (12-29%) suggesting a possible feed-back mechanism for PAP control.
Dillsun (0.5-2 ml/day) did not affect PAP activity and liver lipids but lowered
serum triacylglycerol (27%) and cholesterol (20%) concentrations at a dose of 2
ml/day. Antum administration also showed similar pattern as dillsun except that
it caused a decrease in the liver lipids as well. Garsin (250-1000 mg/Kg)
inhibited PAP activity (14-22%) with simultanous decrease in the liver and serum
lipids; maximum decrease in serum triacylglycerol and cholesterol were 14% and
21%, respectively. The data demonstrated that the antilipidemic drugs tested
exert their effects, at least in part, through alteration of hepatic PAP
activity.

Phosphatidate phosphohydrolase (PAP, EC 3.1.3.4.) catalyzes desphosphorylation
of phosphatidic acid. Two distinct forms of PAP have been identified in rat
liver (1). The metabolic form is located in cytosol and microsomal fraction,
requires Mg2+ and is a regulatory enzyme in triacylglycerol and phospholipids
biosynthesis (2, 3). The second form of PAP present in the plasma membrane, does
not require Mg2+ and is involved in signal transduction (4). The metabolic form
of PAP in the cytosol is in its active form and can be translocated to the
microsomal membrane, where triacylglycerol synthesis occurs, by certain
regulatory factors such as fatty acids and acyl-CoA, in liver (5-6), heart (7)
and adipose tissue (8). The activity of soluble PAP may also be regulated by
certain hormones such as steroid, glucagon (9) and epinephrine (10).

The rate at which triacylglycerol is secreted into the blood stream in the form
of VLDL, and the rate of its clearance from the blood are factors that determine
lipid levels in the serum. Several drugs have been widely used as antilipidemic
agents (11). The mechanisms by which these compounds affect serum lipid levels,
however, are still a contradictory subject. It has been reported that the
hypolipidemic effects of some drugs such as nicotinic acid, clofibrate and
gemfibrozil are, at least in part, resulted from the inhibition of hepatic PAP
activity in rat (12). Among statins HMG-CoA reductase inhibitors (13, 14),
lovastatin and pravastatin have different effects on PAP activity (15)
contradictory to those reported for other antilipidemic drugs (12).

Several medicinal plants such as garlic (Allium sativum) (16) and anethum (anethum
graveolens) (17, 18) have been used as antilipidemic drugs. The mechanisms of
actions of these drugs, however, are not clarified yet. Three antilipidemic
drugs whose constituents are anethum (Antum and Dillsun) and garlic (Garsin) are
produced by the lranian pharmaceutical industries, but the detailed mechanisms
of their actions have not been studied. Antum is a combination of fumaria,
anethum and chichorium which is distributed by Iran Daruk Co. in the forms of
tablets or granules. Dillsun is a 2% essence of anethum presented by Barij Esans
Co. as drops. Garsin is a powder of garlic as tablets produced by Barij Esans
Co.

In the present study the effects of therapeutic doses of dillsum, garsin, antum,
lovastatin and simvatatin on the metabolic form of PAP activity and on the serum
and liver lipids of rats were investigated.

Male Wistar rats (from Pasteur Institute, Iran) were fed and maintained as
described before (19) except that their food contained 2% cholesterol and 0.5%
cholic acid to induce hyperlipidemia (20). The rats received simvastalin or
lovastatin at daily doses of 20 and 40 mg/Kg through feeding tube for 3 days.
Dillsun and garsin were also given daily at various doses (see lgends) by the
feeding tube for 9 and 30 days, respectively. Antum powder was added to the food
(10%) and the animals fed, ad libitum, for 15 days. Control animals for each
drug were treated the same way except that the drug was omitted from the solvent
or food. The rats were sacrificed by decapitation, the blood samples collected
and each liver was homogenized.

Preparation of liver homogenate

The liver of each rat was perfused with isotonic NaCl to remove the blood using
single passage system and homogenized as described previously (12) except that
the homogenizing soution was 50 mM Tris-HCl buffer pH 7.5 containing 1 mM EDTA
and 0.225 M sucrose. The homogenate was centrifuged at 12000 ?g for 30 min and
the supernatant kept for the enzyme assay.

Assay of PAP activity

The activity of PAP was measured as reported before (12).

Other methods

Protein concentration was determined by the method of Lowry et. al (21).
Cholesterol and triacylglycerol in the serum were measured by commercial
enzymatic kits (Darman kave, Iran).The lipids in the liver samples were first
extracted in chloroform :methanol (2:1) according to Norman?s method (22) and
then measured as serum samples. Student t-test was used for statistial analysis.

Results

The changes in hepatic PAP activity and in serum and liver lipids induced by
injection of rats with lovastation and simvastatin are shown in Table 1. Both
lovastatin and simvastatin stimulated hepatic PAP activity . The increase in the
enzyme activity was concentration dependent being 29% and 43% at (40 mg/Kg) dose
for lovastatin and simvastatin, respectively. The stimulatory effects were
accompanied by the decline in triacylglycerol and cholesterol concentrations of
serum and liver. Lovastatin (40 mg/Kg) decreased triacylglycerol of serum (20%)
and liver (29%), whereas the decrease induced by simvastatin in the same tissues
were 29% and 12%, respectively . The decrease in cholesterol of rats receiving
lovastatin was 37% in serum and 28% in liver. Similar patterns of cholesterol
changes were observed for simvastatin.

The finding that certain antilipidemic drugs such as nicotinic acid, clofibrate
or gemfibrozil exert their effects, at least partly, through affecting hepatic
PAP activity (12) led to the assumption that the activity of this enzyme might
also be altered by other drugs such as those investigated in this study. Statins
are known as HMG-CoA reductase inhibitors (23). In the present study both
lovastatin and simvastatin decreased triacylglycerol and cholesterol
concentrations in both serum and liver . This may be explained by reduced
synthesis of these lipids in the liver and/or decreasing their secretion into
the blood as VLDL. Simvastatin also increases LDL receptors resulting in removal
of LDL and its precursors from the serum. It seemed likely that these compounds
also inhibit PAP activity which is a key enzyme in triacylglycerol synthesis
(1), but surprisingly PAP activity was slightly elevated with simultanous
decrease in triacylglycerol levels in both liver and serum. This may be
explained by several assumptions: 1) Previous reports have shown that the
activity of HMG-CoA reductase upon statin treatment is primarly increased (15,
25). These studies have also shown that both HMG-CoA reductase and PAP exhibit a
co-alteration upon administration of certain drugs such as cholesteramine (15).
Several explanations may be proposed for these findings.

2) A correlation between PAP and HMG-CoA reduetase activities have been
previously reported (15). The increased HMG-CoA reductase, therefore, may
stimulate PAP activity through a compensatory mechanism.

3) A feed back control may increase PAP activity after reduction of serum
triacylglycerol concentration.

4) Statins may affect fatty acid and acyl-CoA metabolism which in turn
stimulates translocation of PAP from cytosol to endoplasmic reticulum where its
activity increases (6).

5) Statins exert some of their effects through β-adrenoceptor activation
resulting in an increase in cAMP concentretion (14) which in turn may stimulate
PAP activity in the experimental conditions as it does for gluose 6-phosphate
dehyrogenase in isolated human hepatocytes (26).

The antilipidemic effect of garsin in both serum and liver (Table 2) may be
attributed to the decrease in hepatic lipid synthesis and VLDL secretion into
the blood which is supported by its inhibitory effect on PAP activity, a key
enzyme in triacylglycerol synthesis. Garsin also contains flavonoid compounds
(27) and in animals fed with this compounds serum triacylglycerol has declined
(28). The later report suggested that garlic (the effective portion of garsin)
may prevent lipid absorption in the intestine and increase its consumption in
the muscles resulting in a decrease in serum lipids. Another compound present in
garlic is allicin which in turn lowers serum cholesterol and triacylglycerol
concentrations through inhibition of the activities of HMG-CoA reductase (28,
29) and PAP (Table 2) in the liver.

The mechanism by which antum lowers cholesterol and triacylglycerol is not well
understood. Triacylgycerol synthesis in not involved in this mechanism since PAP
activity was not affected. The major components present in antum are anethum
(68%), fumaria (5%) and chichoriom (5%). The effects of antum, therefore, may be
attributed primarily to anethum. Previous studies have revealed that anethum
lowers total cholesterol, LDL-cholesterol and increases HDL- cholesterol (18).
The major components of the essence of anethum such as carvon, limonen or
α-phellandrene (30) may be responsible for the anethum effects possibly through
affecting the key enzymes such as HMG-CoA reductase or acyl-CoA carboxylase.
Anethum components may also increase LDL receptors in the liver stimulating
cholesterol clearance from the blood.

Dillsun showed similar results to antum on serum lipids, only at 2 ml/day dose,
but did not change liver lipids. Since the major component of dillsun is anethum
it is likely that the differences observed are associated with the drug doses
applied. Nevertheless, other components in antum such as fumaria and chichorium
may be also accounted for the difference between these two drugs.